The cells of the vascular system are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. In engineering materials for vascular therapies or for delivery of cells, a key challenge is to understand the mechanisms that cells use to sense biophysical cues from their environment to enable more effective therapies. Syndecans are proteoglycans that consist of a protein core modified with heparan sulfate glycosaminoglycan chains. Due to their presence on the cell surface and their interaction with cytoskeletal and focal adhesion associated molecules, cell surface proteoglycans are ideally located to serve as mechanosensors of the cellular microenvironment. Our group recently found that syndecan-1 (SDC1) regulates vascular smooth muscle cell differentiation and the response to shear stress in endothelial cells. We hypothesize that SDC1 is a mechanosensor for substrate-mediated cues and can be targeted to alter endothelial cell- biomaterial interactions to better control cell function on these engineered materials. In Aim 1, we will examine the mechanistic role of SDC1 in regulating vascular mechanosensing of substrate compliance and nanotopology. We will create endothelial cell lines with altered expression or mutation in SDC1 and determine how these alterations affect mechanosensing on engineered substrates with varying compliance and nanotopology. Specifically, we will investigate the ability of SDC1 to regulate mechanically mediated signaling through pathways including the Hippo pathway, TGF-? and Rho-GTPases. In addition, we will examine phenotypic regulation and endothelial-to- mesenchymal transition of endothelial cells on these substrates in the presence of biochemical treatments. In Aim 2, we will develop a novel, FRET-based molecular force biosensor that will enable us to examine the forces that are applied to SDC1 during interactions of live cells with the engineered substrates. This biosensor will enable us to probe the importance of the molecular subdomains of SDC1 in mechanosensing, including the glycosylation sites and the cytoplasmic domains. Together, our findings will contribute to our understanding of cellular mechanotransduction and provide needed knowledge for the development of improved vascular devices and cell delivery scaffolds.